Project Summary The hair follicle (HF) cycle is an excellent model for studying adult stem cell (SC) regulation by the microenvironment or niche, as it involves cyclical bouts of destruction (catagen), rest (telogen) and regrowth (anagen). Signals from the dermal papilla (DP) ? a cluster of mesenchymal HF cells ? are thought to induce a switch from a resting to an activated state in bulge/germ SCs during the telogen-to-anagen transition of the hair cycle. However, due to the lack of direct genetic tools for robust, inducible targeting of the telogen DP for gene ablation, the functional evidence for a role of the DP in SC activation is largely indirect. We also know little about the long-term fate and potential of the adult DP and its lineage relationships to the dermal sheath and fibroblasts. Finally, the precise molecular repertoire of the adult DP during the hair cycle has remained poorly defined, due to the limitations of DP isolation and gene expression analysis in previous studies. We recently established genetic labeling strategies for the specific isolation and molecular characterization of embryonic DP precursors and the DP from growing HFs, making it possible to identify their molecular signatures. We then developed embryonic DP-specific gene targeting tools and explored the role of DP signature genes during HF formation. We have now further established the conditions for the robust and inducible genetic targeting of the telogen DP, thus enabling exploration of the cellular turnover and clonal lineage relationships in the DP along with investigation into the molecular mechanisms underlying DP instruction of HF activation during the telogen- to-anagen transition. In these studies, we will rigorously test the hypothesis that the telogen DP serves as a SC-activating niche during the hair cycle. We will establish Crabp1 as the first robust, inducible genetic driver for Cre-mediated targeting of the telogen DP. We will then use Crabp1iCreER mice to determine, with spatial and temporal precision, the cellular turnover of the DP and its lineage relationship to the neighboring dermal sheath and the skin mesenchyme, through genetic fate mapping and pulse-chase label retention experiments, gaining insight into DP identity and lineage potential. We will use our novel driver to ablate PDGFR? and PDGFR? in the telogen DP, assess SC activation status in the bulge/germ, the impact of the loss of PDGF signaling on hair regeneration, and decipher the molecular mechanisms underlying the PDGF signaling-driven DP functions regulating SC activation. We will isolate pure DP, bulge/germ SCs and related cell types at key hair cycle stages, and systematically define their molecular signatures throughout the hair cycle, with unprecedented cellular resolution and sensitivity of gene expression discovery. We will then investigate the functional role of Nr3c1 and other newly identified DP signature genes, by gene ablation with Crabp1iCreER in the telogen DP. With this work, we will greatly expand our knowledge of SC regulation by these niche cells, which will be essential for the development of treatments involving the regeneration of functional skin, including HFs.